Experimental infection of bulls and cows with bluetongue virus serotype 20

Bluetongue virus serotype 20 (BTV20) was inoculated intradermally and subcutaneously in 4 bulls and by the intrauterine route in 8 nulliparous cows after insemination at oestrus. Viraemia was detected intermittently between 8 and 21 days after inoculation. Virus was isolated from tissue samples of 2 cows and a bull after slaughter at 14 days and from one bull at 28 days. Group reactive and type specific antibodies to BTV20 were demonstrated from 17 to 27 days after infection. No antibodies were detected in the animals slaughtered at 14 days. No clinical signs of disease were seen during the experiment and no gross or histopathological changes referable to BTV20 infection were observed post-mortem. Because of the viraemia and the production of detectable serum antibodies, gametes from these cattle would be excluded from export.     

An inactivated vaccine for the control of bluetongue virus serotype 16 infection in sheep in Italy

Because no suitable products are at the moment available to safely control the spread of BTV-16 in Europe, an inactivated vaccine was produced from the reference field isolate of bluetongue virus serotype 16. One group of six sheep was vaccinated subcutaneously with the inactivated vaccine twice, on days 0 and 28, whereas a second group of eight sheep was inoculated with saline solution and used as mock-vaccinated control animals. Seventy-eight days after the first vaccination, all sheep were inoculated subcutaneously with a suspension containing 10(6.3) TCID(50) of a virulent reference BTV-16 isolate. Apart from a transient inflammatory reaction at the injection site, no adverse effects were reported following vaccination. All vaccinated animals developed high titres (7.3-9.3log(2)(ED50%/50 microl)) of virus-specific neutralising antibodies and were resistant to challenge with BTV-16. Conversely, following challenge, control animals developed hyperthermia and long lasting high-titre viraemia.     

Experimental infection of South American camelids with bluetongue virus serotype 8

Bluetongue (BT) is an infectious, non-contagious disease of wild and domestic ruminants. It is caused by bluetongue virus (BTV) and transmitted by Culicoides biting midges. Since 1998, BT has been emerging throughout Europe, threatening not only the na?ve ruminant population. Historically, South American camelids (SAC) were considered to be resistant to BT disease. However, recent fatalities related to BTV in captive SAC have raised questions about their role in BTV epidemiology. Data on the susceptibility of SAC to experimental infection with BTV serotype 8 (BTV-8) were collected in an animal experiment. Three alpacas (Vicugna pacos) and three llamas (Lama glama) were experimentally infected with BTV-8. They displayed very mild clinical signs. Seroconversion was first measured 6-8 days after infection (dpi) by ELISA, and neutralising antibodies appeared 10-13 dpi. BTV-8 RNA levels in blood were very low, and quickly cleared after seroconversion. However, spleens collected post-mortem were still positive for BTV RNA, over 71 days after the last detection in blood samples. Virus isolation was only possible from blood samples of two alpacas by inoculation of highly sensitive interferon alpha/beta receptor-deficient (IFNAR(-/-)) mice. An in vitro experiment demonstrated that significantly lower amounts of BTV-8 adsorb to SAC blood cells than to bovine blood cells. Although this experiment showed that SAC are generally susceptible to a BTV-8 infection, it indicates that these species play a negligible role in BTV epidemiology.     

Bovine infection with bluetongue virus with special emphasis on European serotype 8

Bluetongue virus (BTV) is an arthropod-borne virus infecting domestic and wild ruminants. Infection in cattle is commonly asymptomatic and characterised by a long viraemia. Associated with the emergence and the recrudescence of BTV serotype 8 (BTV-8) in Northern and Central Europe, remarkable differences have been noticed in the transmission and in the clinical expression of the disease, with cattle showing clinical illness and reproductive disorders such as abortion, stillbirth and fetal abnormalities. Several investigations have already indicated the putative ability of the European BTV-8 strain to cross the bovine placenta and to cause congenital infections. The current epidemiological and pathological findings present an unusual picture of the disease in affected bovines.     

Mortality attributable to bluetongue virus serotype 8 infection in Dutch dairy cows

In 2007, bluetongue virus serotype 8 (BTV-8) re-emerged in the Netherlands and a large number of farmers notified morbidity and mortality associated with BTV-8 to the authorities. All dead cows in the Netherlands are registered in one of the three age classes: newborn calves <3 days, calves 3 days to 1 year, and cows >1 year. These registrations result in a complete data set of dead cattle per herd per day from 2003 until 2007. In this study, the mortality associated with BTV-8 for the Dutch dairy industry was estimated, based on this census data. Default, mortality associated with BTV-8 was estimated for the confirmed notification herds. Moreover, an additional analysis was performed to determine if mortality associated with BTV-8 infection occurred in non-notification herds located in BTV-8 infected compartments. A multivariable population-averaged model with a log link function was used for analyses. Separate analyses were conducted for the three different age groups. Confirmed notification herds had an increased cow mortality rate ratio (MRR) (1.4 (95% CI: 1.2-1.6)); calf MRR (1.3 (95% CI: 1.1-1.4)); and newborn calf MRR (1.2 (95% CI: 1.1-1.3)). Furthermore, in non-notification herds in BTV-8 infected compartments, mortality significantly increased 1.1 times (95% CI: 1.1-1.1) in cows, 1.2 times (95% CI: 1.2-1.2) in calves and 1.1 times (1.1-1.1) in newborn calves compared with BTV-8 non-infected months. Using objective census data over a 5-year period, the MRRs indicated increased mortality associated with BTV-8 infection not only in herds of which the farmer notified clinical signs but also in non-notification herds in infected compartments.     

Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection

Potential vertical transmission of wild-type bluetongue virus serotype 8 (BTV-8) in cattle was explored in this experiment. We demonstrated transplacental transmission of wild-type BTV-8 in one calf and oral infection with BTV-8 in another calf. Following the experimental BTV-8 infection of seven out of fifteen multi-parous cows eight months in gestation, each newborn calf was tested prior to colostrum intake for transplacental transmission of BTV by RRT-PCR. If transplacental transmission was not established the calves were fed colostrum from infected dams or colostrum from non-infected dams spiked with BTV-8 containing blood. One calf from an infected dam was born RRT-PCR positive and BTV-specific antibody (Abs) negative, BTV was isolated from its blood. It was born with clinical signs resembling bluetongue and lived for two days. Its post-mortem tissue suspensions were RRT-PCR positive. Of the seven calves fed colostrum from infected dams, none became infected. Of the six calves fed colostrum from non-infected dams spiked with infected blood, one calf became PCR-positive at day 8 post-partum (dpp), seroconverted 27 days later, and remained RRT-PCR and Abs positive for the duration of the experiment (i.e., 70 dpp). This work demonstrates that transplacental transmission in late gestation and oral infection of the neonate with wild-type BTV-8 is possible in cattle under experimental conditions.     

Susceptibility of in vitro produced hatched bovine blastocysts to infection with bluetongue virus serotype 8

ABSTRACT: Bluetongue virus serotype 8 (BTV-8), which caused an epidemic in ruminants in central Western Europe in 2006 and 2007, seems to differ from other bluetongue serotypes in that it can spread transplacentally and has been associated with an increased incidence of abortion and other reproductive problems. For these reasons, and also because BTV-8 is threatening to spread to other parts of the world, there is a need for more information on the consequences of infection during pregnancy. The aim of the present study was to investigate whether hatched (i.e. zona pellucida-free) in vitro produced bovine blastocysts at 8-9 days post insemination are susceptible to BTV-8 and whether such infection induces cell death as indicated by apoptosis. Exposure of hatched in vitro produced bovine blastocysts for 1 h to a medium containing 103.8 or 104.9 TCID50 of the virus resulted in active viral replication in between 25 and 100% of the cells at 72 h post exposure. The infected blastocysts also showed growth arrest as evidenced by lower total cell numbers and a significant level of cellular apoptosis. We conclude from this in vitro study that some of the reproductive problems that are reported when cattle herds are infected with BTV-8 may be attributed to direct infection of blastocysts and other early-stage embryos in utero.     

Bluetongue disease in Swiss sheep breeds: clinical signs after experimental infection with bluetongue virus serotype 8

Clinical disease of bluetongue (BT) in sheep may differ depending on breed, age and immunity of infected sheep and may also vary between serotype and strain of BT virus (BTV). Since there are no data available on the susceptibility of Swiss sheep breeds for BT, we performed experimental infection of the 4 most common Swiss sheep breeds and the highly susceptible Poll Dorset sheep with the BTV serotype 8 (BTV-8) circulating in Northern Europe since 2006. Clinical signs were assessed regarding severity, localisation, progression and time point of their appearance. The results clearly show that the Swiss sheep breeds investigated were susceptible to BTV-8 infection. They developed moderate, BT-characteristic symptoms, which were similar to those observed in Poll Dorset sheep. Regardless of breed, the majority of infected animals showed fever, swelling of the head as well as erosions of the mouth and subcutaneous haemorrhages.     

Molecular cloning of serogroup- and serotype-specific genome segments from bluetongue virus serotype 11

Genome segments 2, 6, 8, and 9 of bluetongue virus (BTV) serotype 11, coding for P2, NS1, NS2, and P6, respectively, were cloned into pUC 8. Sizes of segment-2 and segment-6 clones indicated partial copies (55% and 80% of full length, respectively), whereas segment 8 and 9 clones represented full-length copies. Northern blot hybridizations of the clones to the 5 United States BTV prototypic serotypes (2, 10, 11, 13, and 17) revealed segment-2 clone to be serotype-specific to BTV-11, whereas segment 6, 8, and 9 clones were able to detect all serotypes to varying degrees. All clones failed to detect the related orbivirus, epizootic hemorrhagic disease virus.     

Immunologic response of sheep to inactivated and virulent bluetongue virus

Humoral and cellular immune responses of sheep to inactivated and virulent bluetongue virus (BTV) were studied. All sheep inoculated with inactivated BTV developed BTV group-specific nonneutralizing antibodies, as determined by agar-gel immunodiffusion. The development of group-specific, nonneutralizing, complement-fixing antibodies was variable and appeared to be dependent on immunizing BTV serotype, sheep breed, and individual variation. Virus-neutralizing antibodies were never detected after inoculation with the inactivated BTV. In vitro lymphocyte stimulation to BTV soluble antigen was observed with cells from all inoculated Warhill sheep and with cells from 1 of 3 inoculated Suffolk cross sheep. Complement-fixation titers did not appear to correlate with the degree of protection observed, ie, duration of postchallenge-exposure viremia. The development of postchallenge-exposure neutralizing antibody titer was inversely correlated to protective immunity. The development of a response to BTV antigen in the lymphocyte-stimulation test associated most closely with protection. Warhill sheep were afforded better protection, by inoculation with inactivated BTV, to live virus challenge exposure than were the Suffolk cross sheep. Approximately 30% of the inoculated Suffolk cross sheep responded to challenge exposure with intensified clinical signs of blue-tongue, compared with the challenge-exposed control sheep of the same breed.     

Comparison of virologic and serologic responses of lambs and calves infected with bluetongue virus serotype 10

Four lambs and 3 calves, seronegative to bluetongue virus (BTV), were inoculated intravenously with a highly plaque-purified strain of BTV Serotype 10. A single calf and lamb served as controls and were inoculated with uninfected cell culture lysate. All BTV-inoculated lambs exhibited mild clinical manifestations of bluetongue, whereas infected calves were asymptomatic. Viremia persisted in BTV-infected lambs for 35-42 days, and for 42-56 days in BTV-infected calves. Neutralizing antibodies were first detected in sera collected at Day 14 post-inoculation (PI) from 2 BTV-infected calves and all 4 infected lambs, and at Day 28 PI in the remaining calf. The appearance of neutralizing antibody in serum did not coincide with clearance of virus from blood; BTV and specific neutralizing antibody coexisted in peripheral blood of infected lambs and calves for as long as 28 days. The sequential development, specificity and intensity of virus protein-specific humoral immune responses of lambs and calves were evaluated by immunoprecipitation of [35S]-labelled proteins in BTV-infected cell lysates by sera collected from inoculated animals at bi-weekly intervals PI. Sera from infected lambs and calves reacted most consistently with BTV structural proteins VP2 and VP7, and nonstructural protein NS2, and less consistently with structural protein VP5, and nonstructural protein NS1. Lambs developed humoral immune responses to individual BTV proteins more rapidly than calves, and one calf had especially weak virus protein-specific humoral immune responses; viremia persisted longer in this calf than any other animal in the study. The clearance of virus from the peripheral blood of BTV-infected lambs and calves is not caused simply by the production of virus-specific neutralizing antibody, however the intensity of humoral immune responses to individual BTV proteins might influence the duration of viremia in different animals.     

Single immunization with an inactivated vaccine protects sheep from Schmallenberg virus infection

The arthropod-borne Schmallenberg virus (SBV), family Orthobunyaviridae, emerged in Europe in 2011. SBV is associated with a mild disease in adult ruminants but fetal malformation after an infection during a critical phase of pregnancy. A number of inactivated vaccines have been developed; their efficacy after two injections was demonstrated. To make the vaccination of sheep more efficient and economic the effect of a single immunization with one of these vaccines was investigated in the present study. Five vaccinated sheep and five additional control sheep were inoculated with SBV three weeks after vaccination and the results of a competitive ELISA, a standard microneutralization test and an SBV-specific real-time RT-PCR confirmed vaccine efficacy by demonstrating complete inhibition of viral replication in immunized animals.     

Genetic variation of bluetongue virus serotype 11 isolated from host (sheep) and vector (Culicoides variipennis) at the same site

Genetic relatedness of 2 strains of bluetongue virus (BTV) serotype 11 that were isolated from the same geographic site--one from host (sheep) and the other from the vector Culicoides variipennis during an enzootic of bluetongue at Bruneau, Idaho, in August 1973--was determined by comparing the oligonucleotide fingerprint analyses of the individual double-stranded RNA segments of the genomes. It was observed that the 2 strains of BTV-11 exhibit considerable differences in their genotypes, the percentage of diversity being different for each of the corresponding RNA species of the 2 strains of BTV-11. These results indicate that more than one genotype of BTV can circulate in juxtaposition in a given geographic site. The observed genotypic diversity might be due to the accumulation of point mutations on specific RNA species or antecedent reassortment of RNA segments between different BTV in nature or both.     

Vesicular stomatitis virus replicon expressing the VP2 outer capsid protein of bluetongue virus serotype 8 induces complete protection of sheep against challenge infection

Bluetongue virus (BTV) is an arthropod-borne pathogen that causes an often fatal, hemorrhagic disease in ruminants. Different BTV serotypes occur throughout many temperate and tropical regions of the world. In 2006, BTV serotype 8 (BTV-8) emerged in Central and Northern Europe for the first time. Although this outbreak was eventually controlled using inactivated virus vaccines, the epidemic caused significant economic losses not only from the disease in livestock but also from trade restrictions. To date, BTV vaccines that allow simple serological discrimination of infected and vaccinated animals (DIVA) have not been approved for use in livestock. In this study, we generated recombinant RNA replicon particles based on single-cycle vesicular stomatitis virus (VSV) vectors. Immunization of sheep with infectious VSV replicon particles expressing the outer capsid VP2 protein of BTV-8 resulted in induction of BTV-8 serotype-specific neutralizing antibodies. After challenge with a virulent BTV-8 strain, the vaccinated animals neither developed signs of disease nor showed viremia. In contrast, immunization of sheep with recombinant VP5 - the second outer capsid protein of BTV - did not confer protection. Discrimination of infected from vaccinated animals was readily achieved using an ELISA for detection of antibodies against the VP7 antigen. These data indicate that VSV replicon particles potentially represent a safe and efficacious vaccine platform with which to control future outbreaks by BTV-8 or other serotypes, especially in previously non-endemic regions where discrimination between vaccinated and infected animals is crucial.     

Possible introduction of epizootic hemorrhagic disease of deer virus (serotype 2) and bluetongue virus (serotype 11) into British Columbia in 1987 and 1988 by infected Culicoides carried on the wind.

Outbreaks of epizootic hemorrhagic disease of deer and of bluetongue began in British Columbia in August and October 1987 respectively and recrudescence of infection by both viruses was detected the following year in August. Weather records for up to 18 days before the initial outbreaks of disease, isolation of virus or seroconversion were examined to determine if the viruses could have been introduced by infected Culicoides carried on the wind. Data on temperature, rainfall, wind speed and direction and pressure together with backward trajectory analysis showed that there were suitable winds which could have introduced Culicoides infected with epizootic hemorrhagic disease of deer virus on 13 August 1987 (14 days before disease was observed), Culicoides infected with bluetongue virus on 1 October 1987 (7 days before virus was isolated and 13 days before disease in sheep) and Culicoides infected with bluetongue or epizootic hemorrhagic disease of deer viruses on 20 July 1988 (15 days before seroconversion was detected). The arrival on 13 August 1987 coincided with the passage of a cold front and rain and that on 1 October 1987 with a fall in temperature and calm winds. The source of the Culicoides before arrival could have been the Okanogan Valley as far south as the junction of the Okanogan and Columbia rivers in Washington, USA. Flight would have been at temperatures of 12.6 degrees C or higher and at heights up to 1.5 km.     

Indoor activity of Culicoides associated with livestock in the bluetongue virus (BTV) affected region of northern France during autumn 2006

In August 2006, bluetongue virus (BTV) was detected in the Netherlands, Belgium, western Germany, Luxembourg and northern France for the first time. Consequently, a longitudinal entomological study was conducted in the affected region of northern France (Ardennes) throughout the autumn of 2006. Data on the spatio-temporal distribution of Culicoides (Diptera: Ceratopogonidae) associated with livestock were collected and an attempt was made to identify the vector(s) involved in BTV transmission by means of virus detection in wild-caught biting midges. Weekly sampling using standardized Onderstepoort-type blacklight traps were performed simultaneously both outdoors and indoors in one BTV-free and three BTV-affected farms between September and December 2006. Culicoides were sorted according to farm, location (outdoors vs. indoors), time point (in weeks), species and physiological stage. BTV detection was conducted by RT-PCR on monospecific pools of non-bloodfed parous female Culicoides. The principal results showed: (i) the absence of the Mediterranean vector, C. imicola, (ii) the relatively low abundance of C. dewulfi and C. pulicaris, (iii) the widespread occurrence and abundance of C. obsoletus/C. scoticus with longevity and behaviour compatible with BTV transmission, and (iv) all Culicoides pools tested for BTV were negative. In France, the very low levels of BTV-8 circulation were probably due to the limited introduction of the virus from affected neighbouring countries, and not due to the absence of local vector populations. A key finding has been the substantiation, for the first time, that Culicoides, and particularly the potential vectors C. obsoletus/C. scoticus and C. dewulfi, can be active at night inside livestock buildings and not only outside, as originally believed. The endophagic tendencies of members of the Obsoletus group are discussed in light of the prolonged period of BTV transmission during the autumn of 2006 and the risk of BTV overwintering and resurgence in the spring of 2007. Overall, there is an urgent need to improve our knowledge on the ecology of local Culicoides species before any clear, effective and reliable recommendations can be provided to the veterinary authorities in terms of prevention and control.     

Infection of bovine fetuses at 120 days' gestation with virulent and avirulent strains of bluetongue virus serotype 11.

Bluetongue virus infection in sheep and cattle during fetal development causes neuropathology. Two strains of bluetongue virus serotype 11 designated as UC-2 and UC-8 have different virulence patterns in newborn mice. These viruses have distinctly different electropherotype patterns on polyacrylamide gel electrophoresis indicating a genetic difference in these two viruses of the same serotype. Four bovine fetuses each were inoculated intramuscularly with either UC-2 or UC-8, and one fetus was inoculated with placebo. The inoculation was made intramuscularly through the uterine wall at 120 days' gestation, and the bovine fetuses were recovered by cesarean section 12 or 20 days after inoculation. Fetal blood was collected for virus isolation and serology. Virus was reisolated from brain, blood, lung and liver. Both strains, UC-2 and UC-8, cause severe lesions in the 120 day fetuses. The encephalomalacic lesions occurred earlier and were more severe in fetuses inoculated with UC-8 as compared to those inoculated with UC-2. The subtle differences observed in the fetuses inoculated with the two different strains suggest that there is a difference in pathogenic potential of the two viruses. These differences do not appear to be completely dependent upon the host species.